The goal of this research is to understand the mechanism by which voltage gated sodium channels (VGSCs) are inactivated and how the malfunction of this regulation leads to disease. This inactivation forms a critical part of action potential initiation in excitable cells. The importance of VGSCs is readily underscored by their ubiquitous nature in tissues, including cardiac myocytes, nerves, skeletal muscle, and by diseases associated with their dysregulation, including cardiac arrhythmias, epileptic seizures, and myotonia. The experiments proposed herein are focused on understanding how extracellular Ca2+ contributes to the regulation of two VGSC isoforms by interacting with the channel C-terminal domain (CTD). It is hypothesized that Ca2+ regulates channel inactivation in an isoform specific manner, possibly via the involvement of accessory proteins, such as calmodulin (CaM), or by indirect methods, involving Ca2 +/calmodulin kinase II (Ca2+/CaMKII), and that mutations which disrupt channel gating alter the conformational dynamics associated with regulation. The specific aims proposed are: (1) to ascertain the mechanism of Ca2+-induced disordered-to-ordered transitions responsible for the regulation of VGSCs;(2) to determine if VGSC regulation can occur by an indirect mechanism, such as Ca2+/CaMKII binding to the CTD;(3) and to determine the molecular basis of CTD mutations known to cause VGSC dysregulation. To test these hypotheses, a series of NMR experiments, including spin-relaxation, chemical shift perturbation, and structure determination, are proposed. NMR is well-suited to address these experimental questions as it provides atomic resolution information about changes in the conformation and dynamics of a system. PUBLIC HEALTH RELEVANCE: The proper regulation of voltage gated sodium channels is critical to the function of excitable cells, such as cardiac myocytes, nerves, and skeletal muscle. The proposed research is directly relevant to understanding the mechanism by which regulation occurs and how the malfunction of this regulation results in a host of diseases associated with these channels.